The German published patent application DE 196 34 873 A1 describes an apparatus and a method for distinguishing at least two types of molecule groups exhibiting different fluorescence, bound to analyte molecules, based on time-resolved fluorescence measurements. A light source for illuminating a sample volume is activated for a time interval T1, then, after a time interval T2 a detector is activated for a time T3. From the variation in time of the detector signals recorded during the time interval T3 it is determined which of the at least two molecule groups is contained in the sample volume.
The U.S. Pat. No. 5,315,993 discloses a probe and an apparatus for monitoring a plurality of parameters in an environment, making use of a luminescence phenomenon. A luminescence means is illuminated with a plurality of excitation light components, the amplitudes of which are modulated over time with set modulation frequencies. The luminescence response comprises a plurality of luminescence light components, which exhibit modulations corresponding to the modulations of the excitation light. Via a Fourier transform spectral data are obtained, which enter model equations, from which, inter alia, the lifetime of individual luminescence light components can be determined.
The German published patent application DE 101 52 994 A1 describes a method for the simultaneous optical determination of pH-value and dissolved oxygen of a predominantly aqueous sample. A single sensor matrix is used, containing at least two indicator dyes which produce at least one distinguishable optical signal for the measurable quantities pH-value and dissolved oxygen. In one disclosed embodiment of the method the pH-value and the dissolved oxygen are determined by measuring the decay time of a fluorescence response of the indicators to a pulse-shaped excitation.
The European patent application EP 0 442 060 A2 relates to a ratiometric luminescence measurement for determining a variable, for example the concentration of a substance. A first luminescent material with a first absorption band and a second luminescent material with a second absorption band are used; the first and the second absorption band do not overlap completely. In alternating first and second illumination intervals the luminescent materials are illuminated with a first excitation light within the first, but outside the second absorption band, and with a second excitation light within the second, but outside the first absorption band. The luminescence responses of the first and of the second luminescent material, correspondingly detected during respective first and second response intervals, are evaluated and are used for determining the variable.
The article “Luminescence Lifetime Imaging of Oxygen, pH, and Carbon Dioxide Distribution Using Optical Sensors” by G. Liebsch, I. Klimant, B. Frank, G. Hoist, and O. S. Wolfbeis in Applied Spectroscopy 54, No. 4 (2000), pages 548 to 559, describes the determination of various variables for samples in the wells of a microtiter plate via the dependence of the fluorescence lifetime of materials used as sensors on the respective variable. The fluorescence lifetime is determined as follows: the fluorescence is excited by a light pulse, after the end of which, during each of two intervals with a gap in between and preferentially of equal duration, the fluorescence response of the sensors is integrated. The fluorescence lifetime is determined from the quotient of the values of the integrals obtained in this way. In comparison with methods based on intensity only, this ratiometric method, based on a quotient of measured quantities, has the advantage of being practically independent of the local absolute values of the excitation energy.
The article “Fluorescent Imaging of pH with Optical Sensors Using Time Domain Dual Lifetime Referencing” by G. Liebsch, I. Klimant, C. Krause, and O. S. Wolfbeis in Analytical Chemistry Vol. 73, No. 17, Sep. 1, 2001, pages 4354 to 4363, relates to the determination of the pH-distribution in microtiter plates and on a surface. A combination of two luminescent materials, where the ratio of the amounts of the materials is fixed, is used: a fluorescent material, the fluorescence decay time of which depends on the pH-value, and a phosphorescent material, the phosphorescence decay-time of which is independent of the pH-value. The luminescent materials are excited by illumination, and during the excitation, within a first interval, the combined fluorescence and phosphorescence response of the materials is integrated. Immediately after the end of the excitation the recording of the luminescence response of the materials is interrupted for a period of time which is long enough for the fluorescence to decay practically completely. Afterwards, during a second time interval, which preferentially is of equal length as the first interval, the phosphorescence response of the phosphorescent material is integrated. From the quotient of the two values of the integrals eventually the pH-value can be inferred.
Luminescence-based measuring methods are known for the detection and the quantitative determination of many analytes. If the method is based on the intensity of the luminescence phenomenon, a reproducible illumination of the sample studied, in case of the illumination of an area for an extended sample also the spatial homogeneity of the illumination, is crucial. Other methods are based on the decay time of the luminescence phenomenon and exploit the fact that this decay time in case of numerous luminescent materials depends on specific variables of the environment; examples of such variables are pH-value, concentration of a substance, or temperature. With these methods, for which the prior art cited above contains examples, the luminescence response of a substance used as a sensor material is integrated over defined time intervals, and a ratio of the values of the integrals thus obtained is formed. By this formation of a quotient, due to which the methods are classified as ratiometric, the dependence on fluctuations of the illumination is considerably reduced. With these methods it is not necessarily the decay time or relaxation time of the luminescence phenomenon which is determined explicitly, but instead often a parameter which depends on the relaxation time, for example the quotient of the mentioned values of integrals. If a respective variable to be determined is calibrated against a corresponding respective parameter, the value of the variable can be found from the luminescence response. A difficulty with these methods, however, is to implement the defined time intervals for the integration of the luminescence response with sufficient precision in the measurement apparatus. This involves a certain technical effort implying corresponding costs. Furthermore the technology used is very sensitive, which makes its use, in particular for portable devices in the field, problematic, in particular again with respect to costs.